Mobile genetic elements from the maternal microbiome shape infant gut microbial assembly and metabolism.

The perinatal period represents a critical window for cognitive and immune system development, promoted by maternal and infant gut microbiomes and their metabolites. Here, we tracked the co-development of microbiomes and metabolomes from late pregnancy to 1 year of age using longitudinal multi-omics data from a cohort of 70 mother-infant dyads. We discovered large-scale mother-to-infant interspecies transfer of mobile genetic elements, frequently involving genes associated with diet-related adaptations. Infant gut metabolomes were less diverse than maternal but featured hundreds of unique metabolites and microbe-metabolite associations not detected in mothers. Metabolomes and serum cytokine signatures of infants who received regular-but not extensively hydrolyzed-formula were distinct from those of exclusively breastfed infants. Taken together, our integrative analysis expands the concept of vertical transmission of the gut microbiome and provides original insights into the development of maternal and infant microbiomes and metabolomes during late pregnancy and early life.

Infant Feeding, Gut Permeability, and Gut Inflammation Markers.

OBJECTIVES: Increased gut permeability and gut inflammation have been linked to the development of type 1 diabetes. Little is known on whether and how intake of different foods is linked to these mechanisms in infancy. We investigated whether the amount of breast milk and intake of other foods are associated with gut inflammation marker concentrations and permeability. METHODS: Seventy-three infants were followed from birth to 12 months of age. Their diet was assessed with structured questionnaires and 3-day weighed food records at the age of 3, 6, 9, and 12 months. Gut permeability was assessed with the lactulose/mannitol test and fecal calprotectin and human ß-defensin-2 (HBD-2) concentrations were analyzed from stool samples at the age of 3, 6, 9, and 12 months. The associations between foods and gut inflammation marker concentrations and permeability were analyzed using generalized estimating equations. RESULTS: Gut permeability and gut inflammation marker concentrations decreased during the first year of life. Intake of hydrolyzed infant formula ( P = 0.003) and intake of fruits and juices ( P = 0.001) were associated with lower intestinal permeability. Intake of fruits and juices ( P < 0.001), vegetables ( P < 0.001), and oats ( P = 0.003) were associated with lower concentrations of HBD-2. Higher intake of breast milk was associated with higher fecal calprotectin concentrations ( P < 0.001), while intake of fruits and juices ( P < 0.001), vegetables ( P < 0.001), and potatoes ( P = 0.007) were associated with lower calprotectin concentrations. CONCLUSIONS: Higher intake of breast milk may contribute to higher calprotectin concentration, whereas several complementary foods may decrease gut permeability and concentrations of calprotectin and HBD-2 in infant gut.

Effect of Early Feeding on Intestinal Permeability and Inflammation Markers in Infants with Genetic Susceptibility to Type 1 Diabetes: A Randomized Clinical Trial.

OBJECTIVES: To assess whether weaning to an extensively hydrolyzed formula (EHF) decreases gut permeability and/or markers of intestinal inflammation in infants with HLA-conferred diabetes susceptibility, when compared with conventional formula. STUDY DESIGN: By analyzing 1468 expecting biological parent pairs for HLA-conferred susceptibility for type 1 diabetes, 465 couples (32 %) potentially eligible for the study were identified. After further parental consent, 332 babies to be born were randomized at 35th gestational week. HLA genotyping was performed at birth in 309 infants. Out of 87 eligible children, 73 infants participated in the intervention study: 33 in the EHF group and 40 in the control group. Clinical visits took place at 3, 6, 9, and 12 months of age. The infants were provided either EHF or conventional formula whenever breastfeeding was not available or additional feeding was required over the first 9 months of life. The main outcome was the lactulose to mannitol ratio (L/M ratio) at 9 months. The secondary outcomes were L/M ratio at 3, 6, and 12 months of age, and fecal calprotectin and human beta-defensin 2 (HBD-2) levels at each visit. RESULTS: Compared with controls, the median L/M ratio was lower in the EHF group at 9 months (.006 vs .028; P = .005). Otherwise, the levels of intestinal permeability, fecal calprotectin, and HBD-2 were comparable between the two groups, although slight differences in the age-related dynamics of these markers were observed. CONCLUSIONS: It is possible to decrease intestinal permeability in infancy through weaning to an extensively hydrolyzed formula. This may reduce the early exposure to dietary antigens. TRIAL REGISTRATION: Clinicaltrials.gov: NCT01735123.

Serum proteomics of mother-infant dyads carrying HLA-conferred type 1 diabetes risk.

In-utero and dietary factors make important contributions toward health and development in early childhood. In this respect, serum proteomics of maturing infants can provide insights into studies of childhood diseases, which together with perinatal proteomes could reveal further biological perspectives. Accordingly, to determine differences between feeding groups and changes in infancy, serum proteomics analyses of mother-infant dyads with HLA-conferred susceptibility to type 1 diabetes (n = 22), weaned to either an extensively hydrolyzed or regular cow's milk formula, were made. The LC-MS/MS analyses included samples from the beginning of third trimester, the time of delivery, 3 months postpartum, cord blood, and samples from the infants at 3, 6, 9, and 12 months. Correlations between ranked protein intensities were detected within the dyads, together with perinatal and age-related changes. Comparison with intestinal permeability data revealed a number of significant correlations, which could merit further consideration in this context.

Impact of Extensively Hydrolyzed Infant Formula on Circulating Lipids During Early Life.

Background: Current evidence suggests that the composition of infant formula (IF) affects the gut microbiome, intestinal function, and immune responses during infancy. However, the impact of IF on circulating lipid profiles in infants is still poorly understood. The objectives of this study were to (1) investigate how extensively hydrolyzed IF impacts serum lipidome compared to conventional formula and (2) to associate changes in circulatory lipids with gastrointestinal biomarkers including intestinal permeability. Methods: In a randomized, double-blind controlled nutritional intervention study (n = 73), we applied mass spectrometry-based lipidomics to analyze serum lipids in infants who were fed extensively hydrolyzed formula (HF) or conventional, regular formula (RF). Serum samples were collected at 3, 9, and 12 months of age. Child's growth (weight and length) and intestinal functional markers, including lactulose mannitol (LM) ratio, fecal calprotectin, and fecal beta-defensin, were also measured at given time points. At 3 months of age, stool samples were analyzed by shotgun metagenomics. Results: Concentrations of sphingomyelins were higher in the HF group as compared to the RF group. Triacylglycerols (TGs) containing saturated and monounsaturated fatty acyl chains were found in higher levels in the HF group at 3 months, but downregulated at 9 and 12 months of age. LM ratio was lower in the HF group at 9 months of age. In the RF group, the LM ratio was positively associated with ether-linked lipids. Such an association was, however, not observed in the HF group. Conclusion: Our study suggests that HF intervention changes the circulating lipidome, including those lipids previously found to be associated with progression to islet autoimmunity or overt T1D. Clinical Trial Registration: [Clinicaltrials.gov], identifier [NCT01735123].

Immunomodulatory Effects of Rhinovirus and Enterovirus Infections During the First Year of Life.

Early childhood infections have been implicated in the development of immune-mediated diseases, such as allergies, asthma, and type 1 diabetes. We set out to investigate the immunomodulatory effects of early viral infections experienced before the age of one year on the peripheral regulatory T cell population (Treg) and circulating cytokines in a birth-cohort study of Estonian and Finnish infants. We show here a temporal association of virus infection with the expression of FOXP3 in regulatory T cells. Infants with rhinovirus infection during the preceding 30 days had a higher FOXP3 expression in Treg cells and decreased levels of several cytokines related to Th1 and Th2 responses in comparison to the children without infections. In contrast, FOXP3 expression was significantly decreased in highly activated (CD4+CD127-/loCD25+FOXP3high) regulatory T cells (TregFOXP3high) in the infants who had enterovirus infection during the preceding 30 or 60 days. After enterovirus infections, the cytokine profile showed an upregulation of Th1- and Th17-related cytokines and a decreased activation of CCL22, which is a chemokine derived from dendritic cells and associated with Th2 deviation. Our results reveal that immunoregulatory mechanisms are up-regulated after rhinovirus infections, while enterovirus infections are associated with activation of proinflammatory pathways and decreased immune regulation.

Fungal Dysbiosis and Intestinal Inflammation in Children With Beta-Cell Autoimmunity.

Although gut bacterial dysbiosis is recognized as a regulator of beta-cell autoimmunity, no data is available on fungal dysbiosis in the children at the risk of type 1 diabetes (T1D). We hypothesized that the co-occurrence of fungal and bacterial dysbiosis contributes to the intestinal inflammation and autoimmune destruction of insulin-producing beta-cells in T1D. Fecal and blood samples were collected from 26 children tested positive for at least one diabetes-associated autoantibody (IAA, GADA, IA-2A or ICA) and matched autoantibody-negative children with HLA-conferred susceptibility to T1D (matched for HLA-DQB1 haplotype, age, gender and early childhood nutrition). Bacterial 16S and fungal ITS2 sequencing, and analyses of the markers of intestinal inflammation, namely fecal human beta-defensin-2 (HBD2), calprotectin and secretory total IgA, were performed. Anti-Saccharomyces cerevisiae antibodies (ASCA) and circulating cytokines, IFNG, IL-17 and IL-22, were studied. After these analyses, the children were followed for development of clinical T1D (median 8 years and 8 months). Nine autoantibody positive children were diagnosed with T1D, whereas none of the autoantibody negative children developed T1D during the follow-up. Fungal dysbiosis, characterized by high abundance of fecal Saccharomyces and Candida, was found in the progressors, i.e., children with beta-cell autoimmunity who during the follow-up progressed to clinical T1D. These children showed also bacterial dysbiosis, i.e., increased Bacteroidales and Clostridiales ratio, which was, however, found also in the non-progressors, and is thus a common nominator in the children with beta-cell autoimmunity. Furthermore, the progressors showed markers of intestinal inflammation detected as increased levels of fecal HBD2 and ASCA IgG to fungal antigens. We conclude that the fungal and bacterial dysbiosis, and intestinal inflammation are associated with the development of T1D in children with beta-cell autoimmunity.

Maturation of Gut Microbiota and Circulating Regulatory T Cells and Development of IgE Sensitization in Early Life.

Recent studies suggest that the cross-talk between the gut microbiota and human immune system during the first year of life is an important regulator of the later development of atopic diseases. We explored the changes in the gut microbiota, blood regulatory T cells, and atopic sensitization in a birth-cohort of Estonian and Finnish children followed from 3 to 36 months of age. We describe here an infant Treg phenotype characterized by high Treg frequency, the maturation of Treg population characterized by a decrease in their frequency accompanied with an increase in the highly activated Treg cells. These changes in Treg population associated first with the relative abundance of Bifidobacterium longum followed by increasing colonization with butyrate producing bacteria. High bifidobacterial abundance in the neonatal microbiota appeared to be protective, while colonization with Bacteroides and E. coli was associated with later risk of allergy. Estonian children with lower risk of IgE mediated allergic diseases than Finnish children showed an earlier maturation of the gut microbiota, detected as earlier switch to an increasing abundance of butyrate-producing bacteria, combined with an earlier maturation of Treg cell phenotype and total IgE production. The children with established allergic diseases by age 3 showed a decreased abundance of butyrate producing Faecalibacterium. These results suggest that as well as the maintenance of a bifidobacterial dominated gut microbiota is important during the first weeks of life, the overtake by butyrate producing bacteria seems to be a beneficial shift, which should not be postponed.

Incipient angiogenesis in Barrett's epithelium and lymphangiogenesis in Barrett's adenocarcinoma.

PURPOSE: Barrett's esophagus (BE), a precancerous condition for Barrett's adenocarcinoma, is classically characterized by flames of salmon-colored mucosa extending into normal pale esophageal mucosa. This flaming is thought to be a consequence of continuous erosis of mucosa caused by chronic reflux. Another characteristic feature of Barrett's adenocarcinoma patients is the frequent development of lymph node metastases. We addressed whether onset of angiogenesis occurs in BE and if the lymphatic system might provide a route for Barrett's adenocarcinoma cells to infiltrate regular lymph nodes. PATIENTS AND METHODS: Fifteen surgically resected Barrett's dysplasia or adenocarcinoma patients were included. Immunohistochemistry and a modified whole mount analysis were used. RESULTS: The incipient angiogenesis originates from the pre-existing vascular network in the lamina propria and infiltrates Barrett's epithelium, giving its ominous salmon-red color. Barrett's epithelium-specific goblet cells express vascular endothelial growth factor (VEGF)-A. The immature blood vessels show a relative absence of smooth muscle actin (SMA)-positive mural cells and express VEGF receptor (VEGFR)-2 and matrix metalloproteinase (MMP)-9 on their exterior. Coexpression of VEGF-C and its receptor VEGFR-3 on lymphatic vessels is demonstrated. CONCLUSION: BE is strongly neovascularized not eroded. This novel concept of a molecular mechanism of the origin of BE might emphasize why precancerous BE can give rise to the more cancerous dysplasia and Barrett's adenocarcinoma stages. In addition, adenocarcinoma cells induce lymphangiogenesis. The new lymphangiogenic vessels might provide a systemic route for adenocarcinoma cells to invade circulation and induce lymph node metastasis.

Simultaneous progression of oxidative stress and angiogenesis in malignant transformation of Barrett esophagus.

BACKGROUND: Oxidative stress and angiogenesis are important elements in the pathogenesis of inflammatory diseases and cancer. Our aim was to evaluate the role of both and of antioxidant capacity in the metaplasia-dysplasia-adenocarcinoma sequence in Barrett epithelium. METHODS: In mucosal specimens from 59 patients grouped as having symptomatic gastroesophageal reflux disease, Barrett epithelium, or adenocarcinoma in the esophagus, plus controls, we measured myeloperoxidase activity, superoxidase dismutase activity, glutathione content, and total aromatic DNA adducts. To evaluate blood vessel densities and angioarchitecture, we used immunohistochemistry and a modified whole-mount technique. Sections were stained with endothelium-specific markers and smooth muscle cell actin. RESULTS: The reflux disease-metaplasia-carcinoma sequence revealed progressively increased oxidative stress (increased myeloperoxidase activity), decreased antioxidant capacity (glutathione content), and simultaneous formation of DNA adducts. Pooled data show a negative correlation between glutathione content and DNA adducts (-0.28; P =.05). This sequence was also characterized by increased intensity in microvessels and an increasing percentage of immature blood vessels. In addition, the whole-mount technique offered 3-dimensional evidence that the rich new vascular bed is highly abnormal, with repeated twists, bends, or turns, even in nonmalignant Barrett esophagus. CONCLUSIONS: Increased oxidative stress, decreased antioxidant capacity, and a negative correlation between glutathione content and DNA adduct formation indicate a link between oxidative stress and malignant transformation of Barrett epithelium. Simultaneously, this transformation acquires angiogenic capacity, strong neovascularization, and abnormal angioarchitecture.

Fecal microbiota composition differs between children with ß-cell autoimmunity and those without.

The role of the intestinal microbiota as a regulator of autoimmune diabetes in animal models is well-established, but data on human type 1 diabetes are tentative and based on studies including only a few study subjects. To exclude secondary effects of diabetes and HLA risk genotype on gut microbiota, we compared the intestinal microbiota composition in children with at least two diabetes-associated autoantibodies (n = 18) with autoantibody-negative children matched for age, sex, early feeding history, and HLA risk genotype using pyrosequencing. Principal component analysis indicated that a low abundance of lactate-producing and butyrate-producing species was associated with ß-cell autoimmunity. In addition, a dearth of the two most dominant Bifidobacterium species, Bifidobacterium adolescentis and Bifidobacterium pseudocatenulatum, and an increased abundance of the Bacteroides genus were observed in the children with ß-cell autoimmunity. We did not find increased fecal calprotectin or IgA as marker of inflammation in children with ß-cell autoimmunity. Functional studies related to the observed alterations in the gut microbiome are warranted because the low abundance of bifidobacteria and butyrate-producing species could adversely affect the intestinal epithelial barrier function and inflammation, whereas the apparent importance of the Bacteroides genus in development of type 1 diabetes is insufficiently understood.

Removal of Bovine Insulin From Cow's Milk Formula and Early Initiation of Beta-Cell Autoimmunity in the FINDIA Pilot Study.

OBJECTIVE To test whether weaning to a bovine insulin-free cow's milk formula (CMF) reduces type 1 diabetes mellitus-associated autoantibodies in children at genetic risk. DESIGN Randomized, double-blind pilot trial (Finnish Dietary Intervention Trial for the Prevention of Type 1 Diabetes [FINDIA]). SETTING Three pediatric hospitals in Finland from May 15, 2002, to November 22, 2005. PARTICIPANTS A total of 1113 infants with HLA-conferred susceptibility to type 1 diabetes were randomly assigned to receive study infant formulas; 908 children provided at least 1 follow-up blood sample (last follow-up, June 2009). INTERVENTION The CMF (n = 389), whey-based hydrolyzed formula (WHF) (n = 350), or whey-based FINDIA formula essentially free of bovine insulin (n = 365) during the first 6 months of life whenever breast milk was not available. MAIN OUTCOME MEASURES Primary outcome was beta-cell autoimmunity monitored at ages 3, 6, and 12 months and then annually until age 3 years. Autoantibodies to insulin, the 65-kDa isoform of glutamic acid decarboxylase, and the tyrosine phosphatase-related IA-2 molecule were screened, and islet cell autoantibodies and autoantibodies to zinc transporter 8 were analyzed in infants whose primary screening test results were positive. RESULTS In the intention-to-treat analysis, 6.3% of children in the CMF group, 4.9% of those in the WHF group, and 2.6% of children in the FINDIA group were positive for at least 1 autoantibody by age 3 years. The odds ratios were 0.75 (95% CI, 0.37-1.54) in the WHF group and 0.39 (0.17-0.91) in the FINDIA group when compared with the CMF group. In the treatment-received analysis, the corresponding odds ratios were 0.81 (95% CI, 0.37-1.76) and 0.23 (0.08-0.69). CONCLUSION In comparison with ordinary CMF, weaning to an insulin-free CMF reduced the cumulative incidence of autoantibodies by age 3 years in children at genetic risk of type 1 diabetes mellitus. TRIAL REGISTRATION clinicaltrials.gov Identifier: NCT01055080.

Intracellular and cell surface localization of a complex between alphaMbeta2 integrin and promatrix metalloproteinase-9 progelatinase in neutrophils.

We have recently demonstrated that promatrix metalloproteinases (proMMPs), particularly proMMP-9, are potent ligands of the leukocyte beta(2) integrins. We studied here the complex formation between proMMP-9 and alpha(M)beta(2), the major MMP and integrin of neutrophils. On resting neutrophils, the proMMP-9/alpha(M)beta(2) complex was primarily detected in intracellular granules, but after cellular activation it became localized to the cell surface, as demonstrated by immunoprecipitation and double immunofluorescence. Further indication of the complex formation was that neutrophils and alpha(M)beta(2)-transfected L cells, but not the wild-type L cells or leukocyte adhesion deficiency cells, bound to immobilized proMMP-9 or its recombinant catalytic domain in a beta(2) integrin-dependent manner. Peptides that bound to the alpha(M) integrin-I domain and inhibited its complex formation with proMMP-9 prevented neutrophil migration in a transendothelial assay in vitro and in a thioglycolate-elicited peritonitis in vivo. These results suggest that the translocating proMMP-9/alpha(M)beta(2) complex may be part of the cell surface machinery guiding neutrophil migration.